This work was supported in part by grants from the NIH R01EY012949, R21EY018885, T32 EY0707133. We thank Darwin Hang for his technical assistance.

Animal Use Statement: Animals were cared for in accordance with guidelines described in Guide for the Care and Use of Laboratory Animals, using protocols approved by the University of Minnesota Institutional Animal Care and Use Committee.
1) Preparation of solutions
<ol>
 <li> Before performing electrophysiological recording, intracellular, extracellular, and enzyme solutions need to be prepared.</li>
 <li> Extracellular solution: mix one bottle (8.8g) of powdered Ames' medium (Sigma) with 1 l of H2O and 1.9 g of sodium bicarbonate (23 mM). At all points the extracellular solution is saturated with 95% O2/5% CO2 gas mixture. Transfer 200 mL to a beaker for retina storage. Place the remaining solution in the perfusion system container for gravity perfusion once retina is mounted.</li>
 <li> Intracellular solution: Intracellular solution should have been made previously and then aliquoted into 500-1000 &mu;L aliquots and stored at -20 &deg;C. Intracellular solution varies depending on the experiment needed, for current clamp recordings shown here we use (in mM): 125 K-gluconate, 2 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES, 2 Na2-ATP, 0.5 mM NaGTP pH to 7.2 with KOH 5. A fluorescent tracer such as 10 &mu;M Alexafluor-594 hydrazide (Invitrogen) can be added to visualize dendrites during the experiment, and neurobiotin (0.3%) can also be included for analysis of cell anatomy following recording. When ready to record, thaw intracellular solution.</li>
 <li> Enzyme solution: Combine 10000 Units Collagenase (Worthington Chemicals) with 83 mg Hyaluronidase (Worthington Chemicals) into 4.150 mL of extracellular solution. Store in 50 &mu;L aliquots at -20&deg;C. Aliquots can be used for several weeks. When ready to treat the retina, dilute aliquot into 500 &mu;L of bubbled extracellular solution.</li>
 <li> Phosphate Buffered Solution (PBS): In 800 mL of H2O add 8 g of NaCl, 0.2 g of KCl, 1.4 g of Na2HPO4 and 0.24 g of KH2PO4. Bring the pH to 7.4 with NaOH. Adjust the volume to 1 L.</li>
 <li> 0.2 M Phosphate Buffer: In 800 ml of H2O add 21.8 g of Na2HPO4 and 6.4 g of NaH2PO4. Bring volume to 1 L.</li>
 <li> 4 % Paraformaldehyde Solution: Heat 50ml H2O to 60&deg;C. Add 4g paraformaldehyde, stir for several minutes. Add few drops of 1M NaOH (about 5 drops), keep stirring until solution is clear. Add 50 ml 0.2 M phosphate buffer into beaker. Check pH using pH strips (~7.4). Filter and cool. </li>
 <li> Blocking solution (10% goat serum, 0.5% TritonX100): In 1.8 ml of PBS add 0.2 ml of goat serum and 10 &mu;L Triton X-100. TritonX-100 serves to permeabilise the tissue.</li>
 <li> Streptavidin solution (5% goat serum, 0.5% TritonX100, 1:500 Streptavidin 594): In 1.9 ml of PBS add 0.1 ml of goat serum, 10 &mu;L Triton X-100 and 4 &mu;L of Streptavidin 594.</li>
</ol>
2) Isolation of retina from mouse
Dissection tools needed: 1 #2 Forceps, 2 #5 Forceps, ophthalmologic scissors
<ol>
 <li> Euthanize mouse according to approved protocols, in this case CO2 asphyxiation and enucleate eyeball by gently inserting an angled #2 forceps behind the eye and lifting out the eyeball.</li>
 <li> Place the eyeball into a 35 mm Petri dish with extracellular solution. Cut the optic nerve and any attached extraocular muscles or connective tissue using ophthalmologic scissors. Cut away the cornea with an ophthalmologic scissor and pull out the lens using #5 forceps. Tear the sclera using a pair of #5 forceps and use the forceps to sever the optic nerve where the retina and sclera meet at the optic disk. Gently peel the retina away from the eyecup. Tip: Do each step on both retinas before moving to next step to minimize time.</li>
 <li> Use forceps to remove vitreous attached to the retina. This can be performed by "grabbing" the transparent vitreous above the retina with forceps. The vitreous, if successfully removed, should be visible as a clear, gelatinous substance on the forceps. Tip: Slicing retinas in half following removal of vitreous will maximize the amount of usable tissue and make mounting in the chamber easier.</li>
 <li> Keep the retinas in oxygenated extracellular solution at room temperature in a dark environment with minimal ambient light until they are ready to be digested. Retinas can be stored in this way for ~6 hours.</li>
</ol>
3) Treatment of retina with enzyme mixture and mounting in recording chamber
<ol>
 <li> Dilute an aliquot of collagenase/hyaluronidase in 500 &mu;L extracellular solution, place solution in a 35 mm Petri dish and transfer the retina.</li>
 <li> Keep the retina-containing Petri dish, covered and in darkness, in a 95% O2/5% CO2 environment for 15 min with gentle agitation. This step aids in digesting any remaining vitreous and allows for easier access to the ganglion cell layer. Tip: for retinas from younger animals with less vitreous or if adverse effects are observed, time can be shortened to ~5 minutes.</li>
 <li> Remove retina from enzyme-containing medium and wash extensively in extracellular solution</li>
 <li> Transfer the retina into recording chamber 6 with the photoreceptor layer facing down. Wick away remaining fluid with either a pipette or tissue and roll out sides of retina to flatten tissue being careful to touch only the edges of the retina, particularly avoiding contact with the ganglion cell layer. Place a platinum ring with nylon mesh over retina to anchor the tissue and immediately fill the chamber with extracellular solution.</li>
 <li> Mount the recording chamber onto the microscope stage, attach tube for gravity perfusion and vacuum suction and attach ground wire.</li>
</ol>
4) Localization of EGFP expressing ganglion cells and light stimulation
<ol>
 <li> Perfuse the retina by gravity with extracellular solution at 1 mL/min or faster rates. Tip: If perfusion rate is too slow, the tissue will not be sufficiently oxygenated and synaptic signaling may be disrupted.</li>
 <li> Fill a recording pipette with intracellular solution and insert it into the pipette holder. Tip: Pipettes with resistances in the range of 3 to 5M&Omega; give the best results.</li>
 <li> Visualize the retina under infrared differential interference contrast (DIC) optics at low magnification (10X objective, NA 0.3) to localize the recording pipette. Infrared light is used to minimize retina exposure to visible light and minimize light adaptation. Move to higher magnification (40X objective, NA 0.8) and bring electrode into view just above tissue.</li>
 <li> At high magnification (40X objective), illuminate the retina with epifluorescence (480 nm for EGFP) and select a fluorescent ganglion cell and mark on computer monitor using tape (Figure 1A).</li>
 <li> Under DIC and infrared illumination, position the recording electrode near the targeted ganglion cell (Figure 1A). Clean area immediately covering cell by applying gentle positive pressure to recording electrode via a 12cc plastic syringe connected to the pipette holder with polyethylene tubing. On the amplifier, zero any voltage offsets. Place electrode onto selected ganglion cell until a dimple appears on cell surface.</li>
 <li> With the amplifier in voltage clamp mode, apply a test pulse and monitor the current passing through the pipette. Apply negative pressure to create seal between electrode and cell. Release negative pressure when cell begins to seal and wait until holding current is indicative of a G&Omega; electrical seal between the cell's membrane and pipette. Apply gentle pulses of negative pressure until capacitive transients appear and then release any application of pressure. Tip: if access (series) resistance is high (>30M&Omega;), this can often be ameliorated by additional, very gentle, pulses of negative pressure.</li>
 <li> Once a stable seal has been reached apply a pulse of negative pressure. After whole cell mode is obtained, allow cell to dark adapt for 5 minutes. Record, in current clamp mode, response to full-field, white light stimulation (Figure 1B).</li>
 <li> Following recording, Alexafluor-594 will have diffused to the dendrites. The tracer can be visualized under epifluorescence at 594 nm and used to determine whether a ganglion cell is On-, Off-, or Bi-stratified by switching between epifluorescence and DIC infrared optics 5.</li>
 <li> If neurobiotin is included in pipette and retina is going to be processed for immunohistochemistry, then pipette must be gently retracted to provide minimal disruption of the membrane.</li>
</ol>
5) Analysis of anatomy of recorded ganglion cells
Filling with neurobiotin allows for more detailed morphological analysis of ganglion cells following recording. Tip: If specific correlations between a recorded ganglion cell and detailed morphology is desired, it may be best to record and fill only one RGC per preparation.
<ol>
 <li> If neurobiotin was included in the intracellular solution then place the retina in paraformaldehyde solution and fix overnight at 4&deg;C.</li>
 <li> Rinse in PBS, at least 2 hours, at least 6 changes. Transfer the retina to a plate containing blocking solution. Leave in blocking solution for 2 hours, room temperature on shaker.</li>
 <li> Replace the blocking solution with the Streptavidin solution. Incubate 2 days at 4&deg;C on shaker. Rinse the retina in PBS, 6 rinses, 20 min each.</li>
 <li> Mount the retina with Vectashield in a glass slide. The retina must be placed with the ganglion cells up. Perform confocal microscopy (Figure 1C).</li>
</ol>
Representative Results
If the retina is healthy and the dissection was successful, individual ganglion cells should be visible under DIC at high magnification (Figure 1A). Unhealthy retinas will have large, granulated nuclei and should be discarded. If the synaptic response of a given ganglion cell is intact, then the cell should respond at light onset or offset with action potentials and, in the case of an intrinsically-photosensitive ganglion cell (Figure 1B) , a sustained depolarization.
<img src="/files/ftp_upload/2367/2367fig1.jpg" alt="Figure 1" /> 
 Figure 1. Localization, recording, and staining of a fluorescently labeled retinal ganglion cell. (A) GFP positive RGC visualized under epifluorescent illumination at 40X magnification (left panel) and under DIC illumination (right panel). (B) Synaptic light response of an M2 intrinsically photosensitive retinal ganglion cell recorded in whole-cell current clamp mode from fluorescently labeled RGC. (C) M1 intrinsically photosensitive retinal ganglion cell that was identified under epifluorescence, targeted for whole-cell recording, filled with neurobiotin, and then processed for immunohistochemistry.

<ol>
	<li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li><li>Schmidt, T. M., Taniguchi, K., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+extrinsic+light+responses+in+melanopsin-expressing+ganglion+cells+during+mouse+development.">Intrinsic and extrinsic light responses in melanopsin-expressing ganglion cells during mouse development.</a> J Neurophysiol. 100, 371-384 (2008).</li><li>Huberman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+identification+of+an+On-Off+direction-selective+retinal+ganglion+cell+subtype+reveals+a+layer-specific+subcortical+map+of+posterior+motion.">Genetic identification of an On-Off direction-selective retinal ganglion cell subtype reveals a layer-specific subcortical map of posterior motion.</a> Neuron. 62, 327-334 (2009).</li><li>Siegert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+address+book+for+retinal+cell+types.">Genetic address book for retinal cell types.</a> Nat Neurosci. 12, 1197-1204 (2009).</li><li>Schmidt, T. M., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+differences+among+intrinsically+photosensitive+retinal+ganglion+cells.">Functional and morphological differences among intrinsically photosensitive retinal ganglion cells.</a> J Neurosci. 29, 476-482 (2009).</li><li>Newman, E. A., Bartosch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+eyecup+preparation+for+the+rat+and+mouse.">An eyecup preparation for the rat and mouse.</a> J Neurosci Methods. 93, 169-175 (1999).</li><li>Haverkamp, S., Inta, D., Monyer, H., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+analysis+of+green+fluorescent+protein+in+retinal+neurons+of+four+transgenic+mouse+lines.">Expression analysis of green fluorescent protein in retinal neurons of four transgenic mouse lines.</a> Neuroscience. 160, 126-139 (2009).</li><li>Zhang, D. Q., Zhou, T. R., McMahon, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+retinal+dopaminergic+neurons+underlying+their+multiple+roles+in+vision.">Functional heterogeneity of retinal dopaminergic neurons underlying their multiple roles in vision.</a> J Neurosci. 27, 692-699 (2007).</li><li>Hu, E. H., Dacheux, R. F., Bloomfield, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flattened+retina-eyecup+preparation+suitable+for+electrophysiological+studies+of+neurons+visualized+with+trans-scleral+infrared+illumination.">A flattened retina-eyecup preparation suitable for electrophysiological studies of neurons visualized with trans-scleral infrared illumination.</a> J Neurosci Methods. 103, 209-216 (2000).</li><li>Wu, S. M., Gao, F., Pang, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+circuitry+mediating+light-evoked+signals+in+dark-adapted+mouse+retina.">Synaptic circuitry mediating light-evoked signals in dark-adapted mouse retina.</a> Vision Res. 44, 3277-3288 (2004).</li></ol>

This technique is applicable to any retinal ganglion cell recordings from an isolated retina. Though in the mouse line used above intrinsically photosensitive, melanopsin-expressing RGCs are labeled with EGFP2, 5, this protocol is readily transferable to other fluorescently labeled RGC lines or can be generalized to record from randomly selected RGCs as well. This technique is particularly valuable because the dendritic arbor of each RGC and its respective input neurons are left entirely intact. Recordings ca...

<table><tbody><tr><td>Ames&amp;rsquo; Medium</td><td>Sigma-Aldrich</td><td>A1420</td><td></td></tr><tr><td>Collagenase</td><td>Worthington Biochemical</td><td>LS005273</td><td></td></tr><tr><td>Hyaluronidase</td><td>Worthington Biochemical</td><td>LS002592</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>AlexaFluor 594 Hydrazyde</td><td>Invitrogen</td><td>A10442</td><td></td></tr><tr><td>Neurobiotin</td><td>Vector Laboratories</td><td>SP1120</td><td></td></tr><tr><td>Electrodes</td><td>Sutter Instrument Co.</td><td>BF120-69-10</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>Ophthalmologic Scissor</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>Streptavidin 594</td><td>Invitrogen</td><td>S32356</td><td></td></tr><tr><td>Vectashield</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Goat Serum</td><td>Jackson ImmunoResearch</td><td>005-000-001</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>19210</td><td></td></tr></tbody></table>

an isolated retinal preparation to record light response from genetically labeled retinal ganglion cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

Watch this Scientific Journal Video about An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells at JoVE.com

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

an-isolated-retinal-preparation-to-record-light-response-from

Research

JoVE Journal

Neuroscience

16.7K Views.  University of Minnesota. This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology. 

Video: An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that 
initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The 
recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca2+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca2+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.
Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the 
photoisomerization of its 11-cis chromophore to all-trans5-7. This regeneration begins with the release of all-trans retinal by 
the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all-
trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is 
then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).
Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca2+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer 
segment Ca2+ changes and response to light8-12 as well as the role of inner segment Ca2+ stores in Ca2+ homeostasis13,14. 
Fluorescent dyes can also be used for measuring Mg2+ concentration15, pH, and as tracers of aqueous and membrane compartments16. 
Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single 
photoreceptor cells17-19.

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Biology

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Bioengineering

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Mouse Retina Isolation and Methanol Treatment for Studying Retinal Ganglion Cells

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in RGCs have a close relationship with numerous retinal degenerative diseases. Whole-mount retinal immunostaining is frequently used in experimental studies on RGCs to evaluate the developmental and pathological conditions of the retina. Under some circumstances, some valuable retina samples, such as those from transgenic mice, may need to be retained for a long period without affecting the morphology or number of RGCs. For credible and reproducible experimental results, using an effective preserving medium is essential. Here, we describe the effect of methanol as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage. In brief, during the isolation process, cold methanol (&#8722;20 &#176;C) is pipetted onto the surface of the retina to help fix the tissues and facilitate their permeability, and then the retinas can be stored in cold methanol (&#8722;20 &#176;C) before being immunostained. This protocol describes the retina isolation workflow and tissue sample storage protocol, which is useful and practical for the investigation of RGCs.

Author Spotlight: Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells  

Methanol can be used as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage, which is useful for the investigation of retinal ganglion cells.

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

Studying Bipolar and Horizontal Neuronal Cells in the Murine Split Retina

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

Author Spotlight: Using the Split Retina Technique for Enhanced Access and Accelerated Experiments 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.